Throughout this application various publications are referred to by author and year of publication. Full citations for these references may be found at the end of the specification. The disclosures of these publications are hereby incorporated by reference in their entirety into the subject application to more fully describe the art to which the subject invention pertains.
The ability to methylate specific DNA residues would be very useful. One aspect of the impact of testing DNA methylation impact on gene expression lies in the ability to measure the effect on gene expression of the myriad and complex DNA methylation patterns that have been observed in cells and tissues derived from humans and other organisms. The ability to methylate specific nucleotide residues of a DNA sequence, and test their functional significance, would help investigators focus on the most salient features of the methylome (the pattern and occurrence of methylation in the genome), on a gene-specific basis, for studies on the underpinnings of inheritance, development, gene regulation, environmental effects on gene regulation, and disease pathogenesis. Means for permitting the functional testing of the effect of different patterns of DNA methylation would be additionally useful for investigators.
Residue-specific DNA methylation in vitro or in vivo has not yet been achieved. Native DNA methylation is not preserved in PCR reactions. There exists a description of a patch DNA methylation strategy for functional testing as described in Curradi et al. (2002). For methylating DNA targets in chromatin (native chromosomes), where functional impact can be measured by measuring native gene or mRNA transcript expression, siRNA directed DNA methylation has been reported in plant and yeast. Kawasaki et al. reported siRNA could also direct DNA methylation in human cells (Nature 431; 211, 2004) but this paper was subsequently retracted (Nature 441; 1176, 2006). There are several papers supporting siRNA directed DNA methylation in human cells, albeit with spread of methylation beyond the target site (Morris et al., (2004); Castanotto et al., (2005); Suzuki et al., (2005)). Zinc finger-DNA methyltransferase fusion also can be used for site-directed DNA methylation in chromatin, but the limitation is that the flanks of the target site must have the zinc finger protein recognition sequences (Meister et al., (2010); Smith et al., (2008); Smith et al., (2007); Li et al., (2007)). The limitations inherent to these technologies (imprecision in target sites methylated by siRNA, and target sequence limitations by Zn finger approaches) have not yet been solved, nor have they been coupled to precise functional in vitro testing of their impact on gene expression.
The present invention addresses this need by providing a technique for methylating specific cytosines of CG dinucleotides (“CpG”) in a given DNA and also measuring the impact of that de novo methylation on gene expression.